Treatment of substrates

ABSTRACT

A method of treating a substrate, which method comprises providing an electrolyte in contact with the substrate and an array of electrodes adjacent the surface and in contact with the electrolyte, and altering the potential of at least one electrode so as to generate an active redox product which modifies the substrate adjacent the at least one electrode, characterized in that the electrolyte is chosen such that the active redox product is quenchable by a second redox product. The method is particularly suitable for the step-wise chemical synthesis of oligomers such as oligonucleotides bound to a surface.

[0001] This invention relates to the treatment of substrates using anelectrochemical method. More particularly, this invention relates to amethod of chemically modifying substrates using an electrochemicalmethod.

[0002] Many devices require a pattern of a specific material on asurface. Semiconductor chips are a well-known example of such devices. Amore recent example of such devices are DNA chips, which comprise anarray of oligonucleotides bound to a solid surface (G. Ramsay, NatureBiotechnology, 1998, vol. 16, 40-44).

[0003] The properties of such devices depend on the nature and thepattern of the materials on the surface. Moreover, improvements toexisting devices is driven on the one hand by demands forminiaturisation and on the other by the need for new types of surfaceswhich combine chemical and physical features. Hence, there is anincreasing need for new methods of fabricating devices which require apattern on its surface.

[0004] Several methods exist for the treatment of specific regions ofsurfaces. One method uses photolithographic technology. Specific regionsof a surface are covered with a photolithographic mask and the exposedregions are modified by exposure to UV light. This method has been usedwidely in the manufacture of semiconductors in which apertures arecreated on the surface of a semiconductor wafer coated with alight-sensitive compound.

[0005] A photolithograpic method has also been employed in themanufacture of DNA chips. In this method, oligonucleotides havingphotolabile protecting groups are bound to a solid surface. A region ofthe surface is covered with a photolithographic mask and the exposedregions of the surface irradiated with UV light. Hence, the photolabileprotecting groups may be removed from the exposed region of the surface(G. Ramsay, Nature Biotechnology, 1998, vol. 16, 40-44).

[0006] WO93/22480 describes a method of treating a surface using anelectrochemical method. In this method, there is provided an electrolyteoverlying a surface and an array of electrodes adjacent the surface. Byaltering the potential of one or more electrodes of the array, thesurface adjacent the one or more electrodes may be modified. Theelectrolyte employed is a solution of triethylamine and sulphuric acidin acetonitrile.

[0007] U.S. Pat. No. 6,093,302 describes an electrochemical method ofplacing a material at a specific location on a substrate. The materialis generated at an electrode and generally reacts with a substanceproximate to the electrode. The use of a buffering or scavengingsolution is described. The buffering or scavenging solution is intendedto improve the resolution of the substrate being treated by reactingwith reagents that move away from the immediate vicinity of theelectrodes. However, a bulk solution containing a buffering orscavenging substance has the disadvantage of quenching not only thosereagents that diffuse away from specific electrodes, but also reagentswhich are intended to react at a substrate adjacent a specificelectrode.

[0008] Schuster et al., Science, 2000, 289, 98-101 describes anothermethod of improving the resolution of an electrochemical method oftreating a surface. Schuster employs a sequence of complicated currentpulses to limit diffusion time.

[0009] It is an object of the present invention to provide an improvedmethod of modifying a substrate using electrochemical means. Inparticular, it is an object of the present invention to provide a methodof modifying a substrate with improved resolution.

[0010] Accordingly, the present invention provides a method ofcontrolling the diffusion of a first redox product generated by a firstelectrode comprising generating a second redox product by a secondelectrode in proximity to said first electrode, the first and secondelectrodes being in contact with an electrolyte, wherein saidelectrolyte is such that the first redox product is quenchable by thesecond redox product.

[0011] Preferably, the second electrode is a counter electrode.

[0012] Preferably, the first redox product is an active redox product,which may be used for modifying a substrate adjacent the electrode.

[0013] Hence, in another aspect, the present invention provides a methodof treating a substrate, which method comprises providing an electrolytein contact with the substrate and one or more electrodes adjacent thesubstrate and in contact with the electrolyte, and altering thepotential of at least one electrode so as to generate an active redoxproduct which modifies the substrate adjacent the at least oneelectrode, characterised in that the electrolyte is such that the activeredox product is quenchable by a second redox product.

[0014] By quenchable, it is meant that the second redox product iscapable of reacting with the first redox product and modifying itsreactivity so that the first redox product does not react in the samemanner as it would in its original form. When the first redox product isan active redox product, the reaction between the active redox productand the second redox product will prevent the active redox product frommodifying a substrate. For example, in the case where the active redoxproduct is an acid, the second redox product may be a base. The reactionbetween the acid and the base quenches the acid and prevents it frommodifying a substrate.

[0015] In a preferred embodiment, the quenching reaction will regenerateone or more of the substances in the electrolyte.

[0016] By active redox product, it is meant any oxidation or reductionproduct which is capable of modifying a substrate. The active redoxproduct may be generated directly by oxidation or reduction of asubstance in the electrolyte. Alternatively, the active redox productmay be generated indirectly by oxidation or reduction of a substance inthe electrolyte followed by one or more subsequent reactions with othersubstance(s) in the electrolyte.

[0017] Generally, the active redox product is generated at the surfaceof an electrode. The active redox product may then modify a substrateadjacent thereto. An acid is a preferred example of an active redoxproduct. An acid may be involved in many types of reaction on asubstrate, for example eliminations, substitutions, rearrangements andchemical etching. Preferably, when the active redox product is an acid,the acid is used to remove an acid labile protecting group from asubstrate.

[0018] Acid labile protecting groups are well known to a person skilledin the art and include, for example, acetals (e.g. methoxymethyl,methylthiomethyl, (2-methoxyethoxy)methyl, benzoyloxymethyl,β-(trimethylsilyl)ethoxymethyl, tetrahydropyranyl, benzylidene,isopropylidene, cyclohexylidene, cyclopentylidene), esters (e.g.benzoyl, benzoyloxycarbonyl, tert-butoxycarbonyl), ethers (e.g. trityl,dimethoxytrityl, tert-butyl) and silyl ethers (e.g.tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl). Preferably, theacid labile protecting group is a trityl or dimethoxytrityl (DMT) ether,which are protecting groups commonly used in the synthesis ofoligonucleotides.

[0019] Likewise, the active redox product may be a base. Bases may beinvolved in many types of reaction on a substrate. For example, a basemay be used to remove a base-labile protecting group.

[0020] Base-labile protecting groups are well known to a person skilledin the art and include, for example, 9-fluorenylmethoxycarbonyl (Fmoc)and cyanoethyl groups.

[0021] Radicals are another example of an active redox product. Radicalsmay be used to initiate radical reactions on a substrate.Electrochemical methods for generating radicals will be well known tothe skilled person. One commonly used method for electrochemicallygenerating a radical is the oxidation of carboxylate anions.

[0022] Halogens are another example of an active redox product. Halogensmay be used in, for example oxidation reactions or addition reactions ona substrate. Halogens may be produced electrochemically by oxidation ofthe corresponding halide ion.

[0023] These and other examples of active redox products will be readilyapparent to the skilled person.

[0024] The method of the present invention is used to treat a substrate.As used herein, the term substrate refers to any material or substancewhich is adjacent the electrode(s) and which may be modified by theactive redox product. The substrate may be separate from theelectrode(s), in which case the substrate is placed adjacent theelectrodes and then removed from the vicinity of the electrode(s) afterthe redox reaction has taken place. Alternatively, the substrate(s) maybe attached to the electrode(s) themselves or attached to the samesurface as the electrode(s). If desired, the substrate(s) may be cleavedfrom the electrode(s), or the same surface, after the redox reaction hastaken place.

[0025] Hence, in one embodiment, the substrate is the surface of amaterial which is separate from and adjacent the electrode(s). Thus, thesubstrate may be the surface of a glass, plastics, solid fibre matrix,metal, semiconductor or gel material. The surface of this material maybe modified directly by the redox reaction. Moreover, in thisembodiment, the surface of the material may have substances attachedthereto. Organic compounds, for example, may be attached to (andoptionally cleaved from) the surface of a material by known methods.Thus, substances attached to the surface of the material may be modifiedby the redox reaction.

[0026] In another embodiment, the substrate is a substance attached tothe same surface as electrode(s), or a substance attached to theelectrode(s) themselves, via a linker group. U.S. Pat. No. 6,093,302describes the latter of these arrangements wherein the substrate isattached to the electrode(s) via linker groups.

[0027] The method of the present invention is similar to the methoddescribed in WO93/22480. However, the method of the present inventiondiffers in the choice of electrolyte. WO93/2240 employs an electrolytewhich is triethylamine and sulphuric acid in acetonitrile. The presentinvention uses an electrolyte in which the active redox product isquenchable by at least one other redox product. An advantage of thiselectrolyte is that it is possible to confine precisely an active redoxproduct to the region immediately surrounding the electrode by which itwas generated.

[0028] In the method described in WO93/22480, the confinement of an acidat a specific region is controlled by variation of the electrodepotential. However, the present inventors have found that afterprolonged electrolysis, the acid is unconfined when the electrolyte istriethylamine and sulphuric acid in acetonitrile. Poor confinement ofacid leads to poor resolution of the substrate being treated. Forexample, protons which diffuse away from the immediate vicinity of theanode may react at the substrate in the zone between electrodes. Theadventitious reaction of diffused protons in this way is undesirablefrom the point of view of obtaining high resolution patternedsubstrates. By choosing an electrolyte in accordance with the presentinvention, the problems of the prior art electrolyte may be avoided. Itis an important feature of the present invention that the electrolyte ischosen such that the active redox product is quenchable by at least oneother redox product.

[0029] The skilled person will be aware of many examples of electrolyteswhich produce an active redox product that is quenchable by anotherredox product.

[0030] An example of such an electrolyte is a combination of I⁻ and S₄O₆²⁻. Oxidation of iodide at the anode produces iodine (an active redoxproduct), while reduction of S₄O₆ ²⁻ at the cathode produces S₂O₃ ²⁻,which may quench the iodine generated at the anode. The reactions in theelectrolyte may be represented as follows:

[0031] Anode: 2I⁻−2e⁻→I₂

[0032] Cathode: S₄O₆ ²⁻+2e⁻→2S₂O₃ ²⁻

[0033] Iodine is quenched by the reaction: 2S₂O₃ ²⁻+I₂→S₄O₆ ²⁻+2I⁻

[0034] Preferably, the active redox product is an acid and the quenchingredox product is an anion, preferably an organic radical anion. Usually,the acid is generated at the anode by oxidation of an alcohol, which maybe any aliphatic or aromatic alcohol. In such electrolytes, thequenching anion is usually generated at the cathode by reduction of asuitable substance. Many substances may be reduced at the cathode toproduce an anion, which may quench the acid formed at the anode. Forexample, dissolved molecular oxygen may be reduced at the cathode,thereby generating O₂ ⁻ and/or O₂ ²⁻.

[0035] An example of an electrolyte that produces suitable redoxproducts is a combination of a ketone and a corresponding alcohol.Oxidation of the alcohol at the anode produces a proton (the activeredox product), while reduction of the ketone at the cathode produces aradical anion which may quench the proton generated at the anode.

[0036] The reactions in the electrolyte may be represented as follows:

[0037] Anode: R¹CH(OH)R²→R¹C(O)R²+2H⁺+2e⁻

[0038] Cathode: R¹C(O)R²+e⁻→[R¹C(O)R²]⁻

[0039] wherein:

[0040] R¹ and R² are independently selected from optionally substitutedC₁ to C₁₅ hydrocarbyl wherein up to three C atoms may optionally bereplaced by N, O and/or S atoms; or

[0041] R¹ and R² together form an optionally substituted C₁ to C₁₅cyclohydrocarbylene wherein up to three C atoms may optionally bereplaced by N, O and/or S atoms.

[0042] Preferably, R¹ and R² are independently selected from optionallysubstituted C₁₋₈ alkyl, C₃₋₈ cycloalkyl or phenyl groups.

[0043] The term “hydrocarbyl” is used herein to refer to monovalentgroups consisting of carbon and hydrogen. Hydrocarbyl groups thusinclude alkyl, alkenyl and alkynyl groups (in both straight and branchedchain forms), cycloalkyl (including polycycloalkyl), cycloalkenyl andaryl groups, and combinations of the foregoing, such as alkylcycloalkyl,alkylpolycycloalkyl, alkylaryl, alkenylaryl, alkynylaryl, cycloalkylaryland cycloalkenylaryl groups.

[0044] The term “hydrocarbylene” is used herein to refer to divalentgroups consisting of carbon and hydrogen. Cyclohydrocarbylene groupsthus include cycloalkylene, cycloalkenylene and arylene groups.

[0045] The term “aryl” is used herein to refer to an aromatic group,such as phenyl, naphthyl or anthracyl. Alternatively, when an aryl grouphas carbon atoms replaced by O, N and/or S, the term aryl refers to aheteroaromatic group, such as pyridyl, pyrrolyl, thienyl, furanylimidazolyl, triazolyl, quinolinyl, isoquinolinyl, oxazolyl orisoxazolyl.

[0046] Where reference is made herein to optionally substituted groups,the substituents are preferably selected from C₁ to C₆ alkyl, C₁ to C₆alkoxy, thio, C₁ to C₆ alkylthio, carboxy, carboxy(C₁ to C₆)alkyl,formyl, C₁ to C₆ alkylcarbonyl, C₁ to C₆ alkylcarbonylalkoxy, nitro,trihalomethyl, hydroxy, C₁ to C₆ alkylhydroxy, hydroxy(C₁ to C₆)alkyl,amino, C₁ to C₆ alkylamino, di(C₁ to C₆ alkyl)amino, aminocarboxy, C₁ toC₆ alkylaminocarboxy, di(C₁ to C₆ alkyl)aminocarboxy, aminocarboxy(C₁ toC₆)alkyl, C₁ to C₆ alkylaminocarboxy(C₁ to C₆)alkyl, di(C₁ to C₆alkyl)aminocarboxy(C₁ to C₆)alkyl, C₁ to C₆ alkylcarbonylamino, C₅ to C₈cycloalkyl, C₅ to C₈ cycloalkyl(C₁ to C₆)alkyl, C₁ to C₆alkylcarbonyl(C₁ to C₆ alkyl)amino, halo, C₁ to C₆ alkylhalo,sulphamoyl, tetrazolyl and cyano.

[0047] As used herein, “halo” or “halogen” refers to iodine, bromine,chlorine or fluorine.

[0048] The nature of R¹ and R² may be varied to change the redoxcharacteristics of the electrolyte. For example, the introduction ofsubstituents on R¹ and R² may change the potential at which oxidation orreduction occurs.

[0049] Preferred examples of ketone/alcohol electrolytes are2-propanone/iso-propanol and benzophenone/benzhydrol, in a suitableorganic solvent.

[0050] Another example of a suitable electrolyte isbenzoquinone/hydroquinone and derivatives thereof. Such electrolytes maybe a combination of:

[0051] wherein R³, R⁴, R⁵ and R⁶ are independently selected from:

[0052] hydrogen, halo, nitro, hydroxyl, thio, nitro, amino,

[0053] optionally substituted C₁ to C₁₅ hydrocarbyl wherein up to threeC atoms may optionally be replaced by N, O and/or S atoms; or

[0054] R³ and R⁴ and/or R⁵ and R⁶ together form an optionallysubstituted C₁ to C₁₅ cyclohydrocarbylene wherein up to three C atomsmay optionally be replaced by N, O and/or S atoms.

[0055] Preferably, R³, R⁴, R⁵ and R⁶ are independently selected fromhydrogen, optionally substituted C₁₋₈ alkyl or R³/R⁴ and R⁵/R⁶ togetherform an optionally substituted C₅-C₂ arylene group, such as phenylene.

[0056] The nature of R³, R⁴, R⁵ and R⁶ may be varied to change the redoxcharacteristics of the electrolyte, for example to alter the precisepotential at which oxidation or reduction occurs. Preferred examples ofelectrolytes based on benzoquinone/hydroquinone derivatives areanthraquinone/anthraquinol and duroquinone/duroquinol, in a suitableorganic solvent.

[0057] In a preferred embodiment, the electrolyte comprises a mixture ofbenzoquinone and hydroquinone in acetonitrile. This mixture provides anactive redox product which is a hydrogen ion. The hydrogen ions(protons) are quenchable by a benzoquinone radical anion.

[0058] Specifically, hydroquinone is oxidised at the anode to producebenzoquinone and protons:

[0059] The protons liberated by the oxidation of hydroquinone are mostlylocalised at the anode and may modify a substrate adjacent thereto. Forexample, the protons may deprotect a substrate bearing an acid labileprotecting group.

[0060] Benzoquinone is reduced at the cathode to produce a benzoquinoneradical anion:

[0061] The benzoquinone radical anion is a relatively stable species insolvents such as acetonitrile. This radical anion quenches anyadventitious protons which escape from the immediate vicinity of theanode, in accordance with the following reaction:

[0062] In this way, the resolution of a region of a substrate beingtreated may be improved by localising the active redox product generatedat an electrode, for example, a proton generated at the anode.

[0063] The electrolytes used in the present invention may comprise anysuitable solvent, such as water, THF (tetrahydrofuran), methanol,ethanol, DMF (dimethylformamide), dichloromethane, diethyl ether, DMSO(dimethylsulfoxide) or acetonitrile. The skilled person will appreciatethat the choice of solvent may influence the kinetics or equilibrium ofthe redox reactions at the electrodes and/or the quenching reaction. Thesolvent may affect the reactivity of a species in solution by, forexample, complex formation, hydrogen bonding, dipole-dipole interactionsor charge delocalisation. Preferably, the solvent is an aprotic solventwhich is able to stabilise a radical anion. Examples of aprotic solventsare dichloromethane, DMF, DMSO, acetonitrile and THF. More preferably,the solvent is acetonitrile.

[0064] In a preferred embodiment, the electrolyte additionally comprisesa conductivity enhancer. A conductivity enhancer is a substance whichincreases the conductivity of the electrolyte. It is desirable toincrease the conductivity of the electrolyte so that electrolysis may beperformed at lower voltages than in the absence of a conductivityenhancer. Any ionic substance which is soluble in the electrolyte issuitable for this purpose. For example, when the electrolyte comprisesan organic solvent such as acetonitrile, a suitable conductivityenhancer may be a tetra(C₁₋₈ alkyl) ammonium salt, such astetrabutylammonium hexafluorophosphate.

[0065] The skilled person will appreciate that a salt in the electrolytemay have effects, other than merely increasing the conductivity of theelectrolyte. A salt may affect the kinetics or equilibrium of thequenching reaction and/or the redox reactions at the electrodes. Thepresence of salt is known to influence electrostatic interactionsbetween charged species in solution. This, in turn, may affectreactivity. For example, when the electrolyte ishydroquinone/benzoquinone in acetonitrile, the addition oftetrabutylammonium hexafluorophosphate was found to modify the extent ofthe quenching reaction, as well as increase conductivity.

[0066] The method of the present invention may be performed using anapparatus as described in WO93/22480. The apparatus described inWO93/22480 comprises an array of electrodes spaced apart on aninsulating surface. The electrodes are deposits of platinum, providedwith electrical connecting means for altering their potentials.

[0067] However, it has been found that the method of the presentinvention is preferably performed using iridium electrodes. Hence, thepresent invention provides an array of electrodes, suitable for use inthe method described herein, comprising a block of electricallyinsulating material having a surface, and deposits of iridium spacedapart in an array on the surface, each deposit being provided withelectrical connecting means for altering its potential.

[0068] An advantage of using iridium is that it is highly conductive andchemically inert. Moreover, iridium does not suffer from degradation atthe high electrical potentials which may be employed using the method ofthe present invention. Previously, platinum had been used as theelectrode material. However, it was found that platinum does not adherewell to materials such as silicon wafers, especially at high electrodepotentials. The internal quenching reaction described herein allows theprolonged use of high electrode potentials without significant loss ofresolution at the substrate being treated. The use of high electrodepotentials necessitated a change in electrode design.

[0069] A number of metals were tested for their suitability aselectrodes including aluminium, silver and gold. However, the presentinventors found, surprisingly, that iridium is an excellent choice ofmaterial for the electrodes. Iridium was found not to suffer fromdegradation during electrolysis and adheres well to materials such asoxidized silicon wafers.

[0070] The block of material on which the array of electrodes is formedmay be made from any suitable material such as an insoluble polymer,ceramic oxides (e.g. alumina) or oxidized silicon wafers. Preferably,the material is an oxidized silicon wafer.

[0071] The array of iridium electrodes may be produced using anysuitable method. In a preferred embodiment, the array of electrodes ismade by a process comprising the steps of:

[0072] (i) providing a silicon wafer having a layer of silicon dioxideon the surface thereof;

[0073] (ii) depositing iridium in a spaced apart array on the silicondioxide surface; and

[0074] (iii) annealing the iridium in air at a temperature in the rangeof 200-500° C.

[0075] In a typical procedure, a positive organic photoresist is appliedto a silicon dioxide layer on a silicon wafer. The photoresist isexposed to UV light through a suitable photomask, revealing areas ofsilicon dioxide. Iridium metal is deposited on the surface of thematerial using an electron-beam gun. Removal of the photoresist layerthen reveals the array of electrodes. Finally, the iridium electrodesare annealed in air to promote adhesion to the wafer surface. Typically,the iridium is annealed at about 350° C. for a period of 15 mins to 3hours, preferably about 1 hour.

[0076] The annealing step is important for adhesion of the iridium tosilicon dioxide. The effect of annealing at a temperature of about 350°C. is surprising, given that iridium has a melting point of 2545° C. Ithas been found that, even with a 50 nm layer of iridium, the annealedelectrodes can withstand scratching with a steel scalpel blade.Moreover, the iridium electrodes are able to withstand the harshchemical environment, high potentials and high current which may be usedin the method of the present invention.

[0077] Preferably, the electrodes are an array of parallel lines spacedapart by less than 0.5 mm. Preferably, the electrodes are spaced apartby 0.1-200 microns, more preferably, 1-100 microns and more preferably10-60 microns.

[0078] Preferably, one or more of the electrodes is used as acounter-electrode. Preferably, the substrate to be modified does notform either an electrode or a counter-electrode, in contrast toconventional methods of treating substrates electrochemically. Hence,the method of the present invention may be similar to the methoddescribed in WO93/22480. Further, the substrate to be treated may be anelectrically insulating surface.

[0079] In a preferred embodiment, the present invention provides amethod of performing several treatments in sequence. Thus, theelectrodes of the array are preferably connected up so that eachtreatment is performed by altering the potential of a chosen set of oneor more electrodes of the array.

[0080] Preferably, in the method of the present invention, the substrateto be treated comprises a substance bound to a solid surface. The solidsurface may be the surface bearing the electrodes. Preferably, the solidsurface is a different surface adjacent the electrodes. The active redoxproduct may be used to produce a chemical modification of the solidbound substance. The skilled person can conceive of many active redoxproducts and corresponding chemical modifications. In a preferredembodiment, the substrate to be treated comprises a substance having anacid-labile protecting group. In this preferred embodiment, thetreatment is performed by connecting at least one electrode of the arrayas an anode at a potential which generates an acid in the electrolyte.The acid thus generated will then remove an acid-labile protecting groupfrom the substance bound to the surface in the region adjacent theanode.

[0081] However, it will be appreciated that the active redox product maybe involved in a variety of chemical reactions at a substrate. Onepotential application is in the electrochemical micromachiningtechnology described by Schuster et al., Science, 2000, 289, 98-101. Thetool described by Schuster may be adapted by using an electrolyte inaccordance with the present invention and surrounding the probe tip witha ring of a counter electrode to prevent diffusion of the redox product.Thus, the present invention may be applied to existing nanoscalepatterning techniques. An acid, for example, may be used in etching ornanofabrication applications, which require removal of a small amount ofa material from a surface.

[0082] Alternatively, an acid may be involved in any organic orinorganic reaction promoted by acid. The skilled person will be aware ofa very large number of potential reactions which could be adapted foruse with present invention. Examples of organic reactions includeepoxide openings, additions to multiple bonds, rearrangements,substitutions (e.g. S_(N)1 substitution of a tertiary alcohol),eliminations, formation of enols with subsequent reactions of the enol,and simple protonation of organic acid salts.

[0083] Equally, when the active redox product is a halogen, the halogenmay be involved in mild oxidations, bleaching a substrate orhalogenations. The active redox product may also be a halide ion, whichmay used in substitution reactions.

[0084] The method of the present invention may also be used in thesynthesis of libraries of small organic compounds bound to a surface(see, for example, Schreiber, Science 2000, 287, 1964-1969). Librariesof small organic compounds are important in the field of drug discovery.The range of reactions to which the present method may be applied meansit is ideally suited to the synthesis of such libraries.

[0085] Preferably, the method of the present invention is used in thestepwise chemical synthesis of oligomers, such as oligonucleotides,polysaccharides and proteins. More preferably, the method of the presentinvention is used in the synthesis of oligonucleotides.

[0086] A method of synthesising a set of oligomers may comprise thesteps of:

[0087] (a) providing a substrate having attached thereto an array ofsubstances having a protecting group, an electrolyte in contact with thesubstrate and an array of electrodes adjacent the substrate and incontact with the electrolyte;

[0088] (b) selectively altering the potential of one or more of theelectrodes so as to generate an active redox product which removes theprotecting group from selected substances;

[0089] (c) coupling a protected monomer with the deprotected substancesformed in step (b); and

[0090] (d) repeating steps (b) and (c), while varying the one or moreelectrodes selected in step in step (b), so as to synthesise a set ofoligomers;

[0091] characterized in that the electrolyte is chosen such that theactive redox product is quenchable by at least one other redox product.

[0092] When the above-described method is used in the synthesis ofoligonucleotides, the active redox product is preferably a proton andthe protecting group is preferably an acid labile protecting group, suchas a trityl or dimethoxytrityl (DMT) group which protects a furanylhydroxyl group. The skilled person will appreciate that this method isparticularly suited to the combinatorial synthesis of DNA chips, asdescribed in WO93/22480.

[0093] The above-described method may also be used in the synthesis ofpeptides. For example, peptides may be synthesised by sequential removalof t-butyloxycarbonyl (Boc) protecting groups from nitrogen atoms usingprotons generated at the anode. Other oligomer syntheses will be readilyapparent to a person skilled in the art.

[0094] The present invention will now be described in more detail withreference to the following Figures in which:

[0095]FIG. 1 shows an apparatus suitable for carrying out the method ofthe present invention;

[0096]FIG. 2 shows schematically how a selected region of a substratemay be modified;

[0097]FIG. 3 shows the effect of varying the time of electrolysis; and

[0098]FIG. 4 shows the effect of removing a cathode from the array ofelectrodes.

[0099] Referring to FIG. 1, an array of electrodes is based on anoxidized high-resistivity silicon wafer (1), the upper surface of whichhas a layer of iridium deposited thereon. Gaps (2) are formed in theiridium layer on the silicon wafer using positive-resistphotolithography, resulting in an array of parallel electrodes (3). Thewidth of each electrode and of each gap is approximately 40 microns. Asilicon wafer (4) is placed over the array of electrodes. The siliconwafer is modified to present a DMT-protected nucleoside at its surface.

[0100] Referring to FIG. 2, part of an array of electrodes and asubstrate are shown. The central electrode is an anode and the two otherelectrodes are cathodes. An electrolyte comprising benzoquinone andhydroquinone in acetonitrile is in contact with the electrodes and thesubstrate to be treated. At the anode, hydroquinone is oxidized, thusgenerating benzoquinone and protons. The majority of protons areconfined at a region of the substrate adjacent the anode. Thus, theconfined protons remove DMT groups from a protected nucleotide moietybound to the substrate. However, some of the protons are able to diffuseinto the zone between the anode and the cathode.

[0101] At the cathode, a benzoquinone radical anion is produced byreduction of benzoquinone. The benzoquinone radical anion is arelatively stable species and is able to diffuse into the zone betweenthe anode and the cathode. The benzoquinone radical anion quenches anyprotons which have diffused into this zone, thereby producinghydroquinone and benzoquinone as shown. Thus, the diffused protons areprevented from reacting at a region of the substrate which is notadjacent an anode. By preventing protons from reacting adventitiously inregions between electrodes, the resolution of the patterned substrate isimproved.

[0102]FIG. 2 also shows subsequent treatment of the substrate followingthe above-described detritylation from a specific region of thesubstrate. The free hydroxyl groups are acetylated under standardconditions and the remainder of the DMT groups removed. The resultingfree hydroxyl groups are treated with a fluorescent dye (Cy5phosphoramidite), which allows imaging of the substrate by confocalmicroscopy. Thus, the resolution of the initial detritylation step maybe conveniently analysed. However, it will be readily apparent that anoligomer may be synthesised on a selected region of the substrate usingthe above-described methodology.

[0103] Referring to FIG. 3, the effect of varying the time ofelectrolysis, at a fixed potential of 1.33 V, is shown using confocalmicroscopy. In this Figure, light regions show fluorescence in regionsof the substrate in which no DMT groups have been removed duringelectrolysis. The remaining DMT groups are subsequently replaced with afluorescent Cy5 dye. The light regions are generally adjacent cathodes.Dark regions are those in which the DMT groups have been removed duringelectrolysis. The resulting free hydroxyl-groups are acetylated with anon-fluorescent acetyl group. The dark regions are generally adjacentanodes.

[0104]FIG. 3 shows that after 2.0 s, the DMT groups are fully removed inthe regions adjacent anodes. Moreover, the resolution of the patternedsubstrate is not changed after 80 s. There are sharply defined stripescorresponding to regions adjacent the anodes and cathodes. Thisdemonstrates that the protons generated during electrolysis are strictlyconfined to regions adjacent the anodes, even after prolongedelectrolysis.

[0105] FIGS. 4(a) and (b) show the dramatic effect of removing a cathodefrom the array of electrodes. The dark regions show regions in which DMTgroups have been removed. The electrode potential is fixed at 1.33 V.Once the central cathode has been removed, protons generated at theanode are allowed to diffuse freely into the central region. Thisclearly demonstrates the confining effect of having a cathode generatinga species which is able to quench protons produced at the anode.

[0106] The method of the present invention is described in more detailin the following Examples.

Experimental Section Electrode Assembly

[0107] Conventional positive resist photolithography was used to produceiridium metal (50 nm thickness) electrodes on oxidized high-resistivitysilicon wafers. The oxidized silicon wafers were coated with a positivephotoresist layer and exposed to UV light through a photomask. The waferwas washed with deionized water, baked at 100° C. for 20 minutes and“descummed” by reactive ion etching. The photomask was chosen to give anarray of 96 parallel electrodes, about 7500 microns long and 40 micronswide. The gap in between adjacent electrodes was about 40 microns.

[0108] Iridium was deposited on the wafer by an electron-beam method.Iridium metal was placed in a crucible in a vacuum evaporator and two orthree wafers were situated approximately 20 cm from the crucible in thevacuum evaporator. The wafers were coated with 50 nm iridium by pumpingthe chamber to 3×10⁶ Torr and heating the metal with an electron-beamgun set to 300 mA at 5 kV for about 3 minutes.

[0109] The photoresist layer was then removed by placing the wafer in anultrasonic acetone bath for 30 minutes, thus revealing an array ofelectrodes. The electrodes were annealed at 350° C. in air for about 1hour to promote adhesion with the wafer substrate, and cleaned byreactive ion etching.

[0110] After the heat annealing and cleaning step, each electrode wasindividually connected by ultrasonic gold wire bonding to a printedcircuit board, where digitally controlled “analog switch” integratedcircuits activate electrodes chosen for a given deblocking step. Currentwas applied as multiple independent operational amplifier-controlledvoltages sources. Parallel low-noise instrumentation amplifier feedbackcircuits continuously measured nanoamp-precision current at each of theelectrodes. A computer programmed specially for this work controlled allvoltages, timing, and electrode switching, and collected currentmeasurement data.

Solid Support Assembly

[0111] Polished silicon dioxide wafers were used as the patternedsubstrate supports. Before electrochemical patterning, the wafer surfacewas functionalized with a linker molecule to which the organic reagentswere attached (Gray, D. E., CaseGreen, S. C., Fell, T. S., Dobson, P. J.& Southern, E. M. Ellipsometric and interferometric characterization ofDNA probes inmmobilized on a combinatorial array. Langmuir 13, 2833-2842(1997)). Wafers were placed in a vacuum furnace chamber, 19.11 involume, with an ampoule containing 5 ml glycidoxypropyltrimethoxysilane.After heating the furnace to 185° C., the ampule was heated to 205° C.and the chamber evacuated to 25-30 mBar. After approximately 2.5 ml ofthe silane had evaporated, the chamber was allowed to cool under vacuum(10⁻³ Torr). A “linker molecule” was attached by immersing theglycidoxypropyltrimethoxysilane-derivatized wafers in a 100% solution ofpolyethylene glycol containing a trace of sulfuric acid. DMT-containingphosphoramidite was then covalently attached to the free hydroxyl on thepolyethylene glycol by conventional oligonucleotide synthesis techniques(Beaucage, S. L. & Iyer, R. P. Advances in the Synthesis ofOligonucleotides by the Phosphoramidite Approach. Tetrahedron 48,2223-2311 (1992)). The wafer substrate thus prepared was cut into 1 cmsquares for use in patterning.

EXAMPLE 1

[0112] The electrode array (as prepared above) was placed at a distanceof 20 microns from the solid support. The solid support was prepared asdescribed above, with a thymidine phosphoramidite attached to thepolyethylene glycol linker molecule. The thymidine phosphoramidite hadits 5′-hydroxy group protected by a DMT group.

[0113] A solution of electrolyte (25 mM hydroquinone/25 mM quinone/25 mMtetrabutylammonium hexafluorophosphate in anhydrous acetonitrile) wasintroduced in the cavity between the electrode array and the solidsupport. Selected anodes were then set at 1.33 V with respect to thecathodes and the voltage maintained for 0.2 to 80 s, as shown in FIG. 3.

[0114] Following electrolysis, the silicon wafer was washed withacetonitrile and acetylated with acetic anhydride using a standardmethod. Only the DMT-deprotected regions of the silicon wafer, havingexposed hydroxyl groups, were acetylated in this step.

[0115] The DMT groups not removed by the electrochemical step were thenremoved by treating the whole substrate with a solution ofdichloroacetic acid in dichloromethane. The hydroxyl groups thus exposedwere coupled to Cy5, a fluorescent dye, using a standard phosporamidatecoupling method so that the pattern produced by the electrochemicalgeneration of acid was revealed by observing the fluorescence of the Cy5in a confocal microscope. This sequence of steps is shown in FIG. 2.

[0116]FIG. 3 shows the effect of increasing the electrolysis time at1.33 V. Once a maximum band width is reached after about 2.0 s, theresolution of the substrate is maintained, even after electrolysis for80.0 s.

EXAMPLE 2

[0117] Example 1 was repeated exactly as described above, with theexception that a voltage of 1.33 V was maintained at selected anodes for16 s. The dramatic effect of removing a cathode was investigated, asshown in FIGS. 4(a) and (b). With the central cathode removed, there isno control of diffused protons. The diffused protons are allowed toflood into the central region and are not localised around the anodes.This is evidenced by the central dark area in FIG. 4(b), which containsno fluorescent groups.

EXAMPLE 3

[0118] The general method described in Example 1 was used in thesynthesis of a 17-mer oligomer on a solid support, with 16electrochemically controlled DMT deprotection steps. The method used wasthe same as Example 1, with the exception that the array of electrodeswas placed at a distance of 40 microns from the surface of thesubstrate.

[0119] A uniform covering of a dimethoxytrityl (DMT) protecteddeoxyadenosine (dA) residue was coupled to the polyethylene glycollinker group on the solid support, using standard phosphoramiditecoupling chemistry.

[0120] Following extensive washing with acetonitrile, the electrolyteused in Example 1 was introduced into the cavity between the electrodearray and the solid support. A potential of 1.33 V was applied to aselected anode for 9 s to remove the DMT groups adjacent thereto. Theanode was flanked by two cathodes.

[0121] Following further washing with acetonitrile, a DMT-protectednucleotide residue was coupled to the exposed hydroxy groups using astandard phosphoramidite coupling. The resulting trivalent phosphoruslinkage was oxidized with iodine to yield a pentavalent phosphoruslinkage and the whole silicon wafer was then washed with acetonitrilefollowed by dichloromethane. The standard coupling and oxidation stepsused in the synthesis of oligonucleotides on solid supports are wellknown in the art. (See, for example, ABI Synthesizer Manual, Section 2:“Chemistry for Automated DNA Synthesis”).

[0122] The procedure was repeated, varying the DMT-protected nucleotideresidue introduced in the phosphoramidite coupling step. Thus, anoligonucleotide was synthesized on the solid support.

[0123] This procedure has been employed on an automated apparatus,controlled by a suitably programmed computer, in the synthesis of two17-mer oligonucleotides: wild type “A” human hemoglobin mRNA and thecorresponding “S” type sickle cell mutant mRNA. The 17-mers were builtup on a defined strip of the solid support in excellent yields. Theskilled person will readily appreciate that this method may be used forthe combinatorial synthesis of DNA chips, as described in WO93/22480.

[0124] It will, of course, be appreciated that this invention has beendescribed by way of example only and that modifications of detail may bemade within the scope of the invention.

1-29. (cancelled)
 30. A method of treating a substrate, which methodcomprises providing an electrolyte in contact with the substrate andelectrodes adjacent the substrate and in contact with the electrolyte,altering the potential of at least one first electrode so as to generatean active redox product which modifies the substrate adjacent the firstelectrode, generating a second redox product by a second electrode inproximity to the first electrode, characterised in that the electrolyteis such that the active redox product is quenchable by the second redoxproduct and in that the substrate to be modified does not form either afirst electrode or a second electrode, and is separate from the firstand second electrodes.
 31. The method of claim 30, wherein theelectrolyte comprises a solvent and wherein neither the active redoxproduct nor the second redox product are formed from the solvent. 32.The method of claim 30, wherein the electrolyte comprises an organicsolvent.
 33. The method of claim 32, wherein the solvent is selectedfrom tetrahydrofuran (THF), methanol, ethanol, dimethylformamide (DMF),dichloromethane, diethyl ether, dimethylsulfoxide (DMSO) oracetonitrile.
 34. The method of claim 30, wherein the quenching reactionregenerates the electrolyte.
 35. The method of claim 30, wherein theactive redox product is a proton.
 36. The method of claim 30, whereinthe second redox product is an organic radical anion.
 37. The method ofclaim 31, wherein the electrolyte is a solution of hydroquinone andbenzoquinone, or derivatives thereof.
 38. The method of claim 30,wherein the electrolyte is a solution of:

wherein R³, R⁴, R⁵ and R⁶ are independently selected from: hydrogen,halo, hydroxyl, thio, nitro, amino, optionally substituted C₁ to C₁₅hydrocarbyl wherein up to three C atoms may optionally be replaced by N,O and/or S atoms; or R³ and R⁴ and/or R⁵ and R⁶ together form anoptionally substituted C₁ and C₁₅ cyclohydrocarbylene wherein up tothree C atoms may optionally be replaced by N, O and/or S atoms.
 39. Themethod of claim 30, wherein the electrolyte is a solution ofhydroquinone and benzoquinone in acetonitrile.
 40. The method of claim30, wherein the electrolyte further comprises a conductivity enhancer.41. The method of claim 40, wherein the conductivity enhancer is atetra(C₁₋₈ alkyl) ammonium salt.
 42. The method of claim 41, wherein theconductivity enhancer is tetrabutylammonium hexafluorophosphate.
 43. Themethod of claim 30, wherein, for the purposes of performing severaltreatments in sequence, an array of electrodes are connected up so thateach treatment is performed by altering the potential of a chosen set ofone or more of the electrodes of the array.
 44. The method of claim 30,wherein the substrate comprises an array of substances bound to asurface.
 45. The method of claim 44, wherein the surface is a surface ofan oxidized silicon wafer.
 46. The method of claim 44, wherein thesubstances to be treated comprise an acid labile protecting group. 47.The method of claim 46, wherein each treatment is performed byconnecting at least one electrode of the array as an anode at apotential to remove an acid labile protecting group from a substance onthe surface.
 48. The method of claim 43, wherein the or each treatmentis performed in the course of a stepwise chemical synthesis of anoligomer.
 49. A method of synthesising a set of oligomers comprising thesteps of: (a) providing a substrate having attached thereto an array ofsubstances having a protecting group, an electrolyte in contact with thesubstrate and an array of electrodes adjacent the substrate and incontact with the electrolyte; (b) selectively altering the potential ofone or more of the electrodes so as to generate (i) an active redoxproduct which removes the protecting group from selected substances anda second redox product; (c) coupling a protected monomer with thedeprotected substances formed in step (b); and (d) repeating steps (b)and (c), while varying the one or more electrodes selected in step (b),so as to synthesise a set of oligomers; characterized in that theelectrolyte is chosen such that the active redox product is quenchableby the second redox product, wherein the substrate to be modified doesnot form an electrode, and is separate from the array of electrodes. 50.The method of claim 49, wherein the electrolyte comprises a solvent andwherein neither the active redox product nor the second redox productare formed from the solvent.
 51. The method of claim 49, wherein theelectrolyte comprises an organic solvent.
 52. The method of claim 51,wherein the solvent is selected from tetrahydrofuran (THF), methanol,ethanol, dimethylformamide (DMF), dichloromethane, diethyl ether,dimethylsulfoxide (DMSO) or acetonitrile.
 53. The method of claim 49,wherein the quenching reaction regenerates the electrolyte.
 54. Themethod of claim 49, wherein the array of substances are attached to asurface and the oligomer is synthesized on said surface.
 55. The methodof claim 49, wherein the oligomers are oligonucleotides.
 56. The methodof claim 49, wherein the active redox product is a proton and theprotecting groups are acid labile protecting groups.
 57. The method ofclaim 49, wherein the electrolyte is a solution of hydroquinone andbenzoquinone, or derivatives thereof.
 58. An array of electrodes,comprising a block of insulating material having a surface, and depositsof iridium spaced apart in an array on the surface, each deposit beingprovided with electrical connecting means for altering its potential.59. The array of claim 58, wherein the block of insulating material isan oxidized silicon wafer.
 60. The array of claim 58, wherein thedeposits of iridium are in the form of spaced apart parallel lines. 61.The array of claim 58, wherein said array is made by a processcomprising the steps of: (i) providing a silicon wafer having a layer ofsilicon dioxide on the surface thereof; (ii) depositing iridium in aspaced apart array on the silicon dioxide surface; and (iii) annealingthe iridium in air at a temperature in the range of 200-500° C.